Inherited retinal dystrophies (IRDs) are a genetically diverse group of vision loss disorders with over 360 implicated genes. However, 30-50% of cases remain unresolved after panel-based clinical testing and may benefit from exome or genome sequencing for a genetic diagnosis. To manage the extensive and analytically demanding datasets generated by genome sequencing, we developed ReDGAP (Retinal Degeneration Genome Analysis Pipeline), a phenotype-guided, semi-automated genome analysis pipeline that integrates clinical phenotyping with flexible variant scoring to prioritize variants of interest (https://github.com/vincentlab-la/ReDGAP). The pipeline supports the joint analysis of multiple variant classes, using an evidence-weighted scoring system informed by in silico predictors. Validation in eleven previously solved IRD cases achieved a 100% re-identification rate. Application to five unsolved cases yielded diagnoses in four (80%), including intronic variants in CRB1 and HGSNAT, a tandem duplication in OAT, and a 5'UTR deletion affecting a retina-specific promoter of RPGRIP1. Functional validation confirmed transcript-level disruptions in three variants, while computational analysis demonstrated regulatory impact in the fourth. Integrating phenotypic data with broad variant analysis offers a tailored model for improving IRD diagnostics, enabling timely molecular diagnoses and informing eligibility for emerging gene-targeted therapies. This positions ReDGAP as a tailored, clinically relevant model for investigating rare diseases within the evolving landscape of precision health.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the aged population with the late stage geographic atrophy (GA). Risk factors for AMD include age, genetic variants in the complement system, nutritional factors, and alterations in the gut microbiome (GM). To identify taxonomic and functional differences in the microbiome associated to disease pathophysiology and genetic risk factors, this study investigated the GM and the ocular surface microbiome (OSM) of GA patients compared to healthy controls by whole-metagenome shotgun sequencing. 16 AMD-associated SNPs were genotyped from blood samples using TaqMan assays and Sanger sequencing. While GA patients showed differences in the GM, and altered metabolic pathways including inosine 5'-phosphate degradation, NAD salvage, and ketogenesis, no alterations in the OSM were found. Genetic analysis associated SNP rs1061170 in the complement factor H gene with GA. These findings suggest that microbial alterations may contribute to GA through inflammation and oxidative stress.Registry: ClinicalTrials.gov, NCT02438111, Registration date: 28 April 2015, and NCT04658238, Registration date: 01 December 2020.

Singapore has advanced in precision medicine, which is largely based on genetic testing and sequencing, yet its safeguard against genetic discrimination (GD) is limited to a non-binding insurance moratorium, with no protections in employment. This study examined the prevalence of self-reported GD and factors influencing willingness to undergo genetic testing in Singapore. A cross-sectional survey assessed experiences of GD, awareness of protections and testing willingness. Twenty percent reported GD in insurance and 9% in employment. The majority identified existing safeguards incorrectly. Sixty-four percent expressed willingness to undergo medically indicated genetic testing. Willingness was positively associated with education, trust in healthcare and perceived fair treatment and negatively associated with age, parental status, deterministic thinking and cultural-religious beliefs. The results highlight that, though policymakers aim to mitigate GD in Singapore, enhanced legal protections and public education are needed to support equitable access to genetic testing.

This study aimed to evaluate the feasibility of whole-genome sequencing (WGS) combined with computational tools for spinal muscular atrophy (SMA) carrier screening and disease diagnosis in Taiwan. WGS data from 1492 Taiwan Biobank participants and two patients with SMA were analysed to determine the SMN1 and SMN2 copy numbers using Illumina DRAGEN SMN Caller and validated by multiplex ligation-dependent probe amplification (MLPA). Among 1480 samples analysed, 23 SMA carriers were identified, yielding a carrier frequency of 1.55%. MLPA confirmed the accuracy of SMN1 and SMN2 copy number results detected using WGS. Both patients with SMA presented compound heterozygous variants with one SMN1 copy loss and the other SMN1 variant, specifically SMN1,c.815A>G, and SMN1,c.81+2_81+3delTG, respectively. Taken together, combining WGS with advanced bioinformatics tools is a feasible and promising approach for SMA carrier screening and disease diagnosis.

RNA sequencing (RNA-seq) has become key to complementing exome and genome sequencing for variant interpretation. We present a minimally invasive RNA-seq protocol using short-term cultured peripheral blood mononuclear cells (PBMCs) with and without cycloheximide treatment, enabling detection of transcripts subject to nonsense-mediated decay. While broadly applicable, this protocol is particularly suited for neurodevelopmental disorders, as up to 80% of the genes in our intellectual disability and epilepsy gene panel are expressed in PBMCs. Applied to 46 affected individuals and 15 parents, RNA-seq revealed splicing defects in six of nine individuals with splice variants, allowing reclassification of seven variants. Targeted cDNA analysis confirmed aberrant splicing in four individuals but missed intron retention in two. Global analyses (FRASER, OUTRIDER, and monoallelic expression) supported findings but did not yield new diagnoses. We propose a flowchart integrating RNA-seq into diagnostic workflows. Overall, our protocol is easily implementable, captures complex splicing events, and enhances variant classification.

To identify novel genetic loci for children refractive error, we performed a meta-analysis of two genome-wide association studies (GWASs) of spherical equivalent (SE) in 1,237 children from the population-based Hong Kong Children Eye Study (HKCES) and the Low Concentration Atropine for Myopia Progression (LAMP) study. Replication was conducted in 4,093 Chinese children and 1,814 Chinese adults. Four lead-SNPs (MIR4275 rs292034, TENM3 rs17074027, LOC101928911 rs6925312 and FAM135B rs4609227) showed genome-wide significant association (P ≤ 5.0 × 10−8) with SE. TENM3 had been associated with myopia in adults before, whilst the other three loci, MIR4275, LOC101928911 and FAM135B, were novel. Significant interaction between genetic risk scores (GRS) and near work on SE was also detected (βinteraction = 0.14, Pinteraction = 0.0003). This study identified novel genetic loci for children refractive error and suggested myopia intervention can be individualized based on the genetic risk of children.

Genetic testing for solid tumor syndromes typically uses peripheral blood leukocytes (PBL) as the source of germline DNA. This approach has shortcomings in certain situations, such as somatic mosaicism and hematologic malignancies. Here we describe a case where germline genetic testing on PBL revealed an unsuspected diagnosis of myelodysplastic syndrome (MDS). A 68-year-old male with a history of three solid tumors and a significant family history of cancer underwent germline genetic testing with a 76-gene hereditary cancer panel. Initial testing using PBL revealed deletions of the entire APC and CTNNA1 genes, suggestive of a contiguous deletion of chromosome 5 (del(5q)). Subsequent testing on cultured fibroblasts was negative, indicating the deletions were somatic. Bone marrow analysis confirmed the presence of del(5q) and a diagnosis of MDS. This case demonstrates the potential to uncover hematologic disorders through hereditary cancer genetic testing, emphasizing the importance of careful results interpretation, multidisciplinary follow-up, and DNA source selection.

Senior-Løken syndrome is a rare ciliopathy characterized by retinal dystrophy and nephronophthisis. This autosomal recessive inherited disease is caused by pathogenic variants in several genes, including IQCB1. We present a Senior-Løken case that remained genetically unexplained after routine genetic testing, including exome and genome sequencing. To identify the genetic cause for this individual, a combination of innovative long-read technologies was employed. Using optical genome mapping, an intronic 6.2-kb insertion in IQCB1 was revealed. Validation by long-read genome sequencing determined that this insertion consisted of a LINE-1/ERV1-mobile element. The variant was found in trans with a pathogenic IQCB1 2-bp deletion previously identified by exome sequencing. To investigate the consequences of the insertion, targeted long-read RNA-sequencing was performed, revealing a complex splice defect causing the introduction of a premature stop codon. This finding suggests that mobile element insertions represent a yet underestimated variant type that is difficult to detect using short-read sequencing.

Familial adenomatous polyposis (FAP) is caused by pathogenic germline variants in the tumor suppressor gene APC. Confirmation of diagnosis was not achieved by cancer gene panel and exome sequencing or custom array-CGH in a family with suspected FAP across five generations. Long-read genome sequencing (PacBio), short-read genome sequencing (Illumina), short-read RNA sequencing, and further validations were performed in different tissues of multiple family members. Long-read genome sequencing resolved a 6 kb full-length intronic insertion of a heterozygous LINE-1 element between exons 7 and 8 of APC that could be detected but not fully resolved by short-read genome sequencing. Targeted RNA analysis revealed aberrant splicing resulting in the formation of a pseudo-exon with a premature stop codon. The variant segregated with the phenotype in several family members allowing its evaluation as likely pathogenic. This study supports the utility of long-read DNA sequencing and complementary RNA approaches to tackle unsolved cases of hereditary disease.

Individuals affected by a rare disease often experience a long and arduous diagnostic odyssey. Delivery of genetic answers in a timely manner is critical to affected individuals and their families. Multi-omics, a term which usually encompasses genomics, transcriptomics, proteomics, metabolomics and lipidomics, has gained increasing popularity in rare disease research and diagnosis over the past decade. Mass spectrometry (MS) is a technique allowing the study of proteins, metabolites and lipids and their fragments at scale, enabling researchers to effectively determine the presence and abundance of thousands of molecules in a single test, accurately quantify their specific levels, identify potential therapeutic biomarkers, detect differentially expressed proteins in patients with rare diseases, and monitor disease progression and treatment response. In this review, we focus on mass spectrometry (MS)-based omics and survey the literature describing the utility of different MS-based omics and how they have transformed rare disease research and diagnosis.